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Blackwell Science, LtdOxford, UKEMIEnvironmental Microbiology 1462-2912Society for Applied Microbiology and Blackwell Publishing Ltd, 200475685697Original ArticleDiversity of ‘Spumella-like’ flagellatesJ. Boenigk, K. Pfandl, P. Stadler and A. Chatzinotas Environmental Microbiology (2005) 7(5), 685–697 doi:10.1111/j.1462-2920.2005.00743.x High diversity of the ‘Spumella-like’ flagellates: an investigation based on the SSU rRNA gene sequences of isolates from habitats located in six different geographic regions Jens Boenigk,1* Karin Pfandl,1 Peter Stadler1 and Antonis Chatzinotas2 1 Institute for Limnology, Austrian Academy of Sciences, Mondseestr. 9, A-5310 Mondsee, Austria. 2 UFZ Centre for Environmental Research Leipzig-Halle, Department of Environmental Microbiology, Permoserstrasse 15, D-04318 Leipzig, Germany. lates and in terms of the number of genotypes, (ii) Spumella and Ochromonas are polyphyletic, and (iii) based on the SSU rRNA gene no biogeographical restriction of certain branches could be observed even though different ecotypes may be represented by the same genotype. Introduction Summary We isolated 28 strains of ‘Spumella-like’ flagellates from different freshwater and soil habitats in Austria, People’s Republic of China, Nepal, New Zealand, Uganda, Kenya, Tanzania and Hawaii by use of a modified filtration–acclimatization method. ‘Spumella-like’ flagellates were found in all of the samples and were often among the dominant bacterivorous flagellates in the respective environments. The small subunit ribosomal RNA (SSU rRNA) gene sequence of the isolates was determined and aligned with previously published sequences of members belonging to the Chrysophyceae sensu stricto. Phylogenetic analysis of the 28 new sequences confirmed their position within the Chrysophyceae sensu stricto and positioned them within different clades. Most of the sequences grouped within clade C and formed several subclusters separated from each other by green taxa including flagellates belonging to Ochromonas, Dinobryon, Poterioochromonas and others. All soil isolates clustered together (subcluster C1) with the soil strain Spumella elongata and the undescribed soil strain ‘Spumella danica’. Aquatic isolates were affiliated with at least two branches (C2 and C3). Sequence similarity to the closest related member of the Chrysophyceae ranged between 92% and 99.6%, sequence divergence among the ‘Spumella-like’ flagellates was as high as 10%. We conclude that (i) the ‘Spumella-like’ flagellates are a diverse group both in terms of sequence dissimilarity between iso- Received 24 June, 2004; accepted 13 October, 2004. *For correspondence. E-mail [email protected]; Tel. (+43) 6232 312529; Fax (+43) 6232 3578. © 2005 Society for Applied Microbiology and Blackwell Publishing Ltd Since 1983 Azam and colleagues (Azam et al., 1983) introduced the concept of the microbial loop, the significance of heterotrophic single-cell eukaryotes for carbon transfer through aquatic food webs has become generally accepted (Wylie and Currie, 1991; Sanders et al., 1994; Sherr and Sherr, 1994; Arndt et al., 2000; Boenigk and Arndt, 2002). The nanoflagellate genera Spumella/Monas are among the most important heterotrophic eukaryotes in many different ecosystems: On annual average 20– 50% of the pelagic heterotrophic nanoflagellate (HNF) biomass in freshwaters is formed by small heterokont taxa, mainly colourless chrysophytes (= chrysomonads) and bicosoecids (Salbrechter and Arndt, 1994; Arndt et al., 2000). Spumella, which represents a typical colourless chrysophyte has been reported to be generally common in freshwaters (Carrick and Fahnenstiel, 1989; Sanders et al., 1989; Bennet et al., 1990; Carrias et al., 1998). Even in benthic sites, colourless chrysophytes make up to 30%, but usually much less (Arndt et al., 2000). In addition, the chrysophyte genera Ochromonas and Poterioochromonas are assumed to be among the dominant mixotrophs (Bennet et al., 1990). The primary mode of nutrition of these mixotrophs often is bacterivory (Andersson et al., 1989). These bacterivorous chrysophytes (family Chromulinaceae sensu; Preisig, 1995) therefore are responsible for a significant part of the bacterivory in freshwater systems and are an important link between bacterial production and higher trophic levels. Thus far, field investigations and food web models have been primarily focused on the so-called ‘functional groups’, i.e. bacteria, heterotrophic nanoflagellates, ciliates, etc. It became evident, however, that such models cannot sufficiently describe the specific interactions and pathways within the microbial food web. For this reason, 686 J. Boenigk, K. Pfandl, P. Stadler and A. Chatzinotas attention is increasingly drawn towards species- or taxonspecific investigations and assessing the diversity of the free-living flagellates is becoming increasingly important (e.g. Kinner et al., 1998; Cleven and Weisse, 2001). The attempt for specific investigations is often hampered by the sparseness of diagnostic characteristics for taxonomic identification. This is valid in particular for the small heterotrophic chrysophytes which are often summed up as ‘Spumella-like flagellates’ or Spumella spp. in many field studies (Weisse, 1997; Auer and Arndt, 2001; Cleven and Weisse, 2001; Weitere and Arndt, 2003). The sparseness of diagnostic features and molecular data does not provide any clarification whether these organisms form either a mono- or a polyphyletic group: Spumella is considered to be the colourless counterpart of Ochromonas (Preisig et al., 1991), but Ochromonas ssp. have already been reported to cluster in different branches, i.e. together with Poterioochromonas, Chrysoxys and Chromulina, and have therefore been suggested to be polyphyletic (Andersen et al., 1999). In addition, occasional loss of colour has been independently described for several species of Ochromonas (Bourrelly, 1957) and for these reasons the separation of Ochromonas and Spumella is doubtful (Fenchel, 1982a,b; Preisig et al., 1991). Similarly, in ecophysiological laboratory investigations, members of the heterotrophic chrysophytes have been widely used as model organisms, but these investigations are based on very few strains (cf. Cowling, 1991) that often lack precise taxonomic identification (Holen and Boraas, 1991; Zwart and Darbyshire, 1992; Rothhaupt, 1997; Boenigk, 2002). In laboratory and field studies, these organisms are usually treated as a black box assuming, basically, similar ecological characteristics. Recent studies revealed, however, that even closely related protist taxa differ in their basic response to environmental factors (Weisse, 2002; Boenigk et al., 2004). It is therefore of urgent interest to survey the diversity and the taxonomic and ecological integrity of the socalled ‘Spumella-like’ flagellates. To minimize confusion resulting from the different botanical (Chrysophyceae) and zoological (Chrysomonadida) nomenclatures we will generally follow the concept of Preisig (1995) throughout our manuscript and use the term Chrysophyceae. We isolated 28 strains of ‘Spumella-like’ flagellates from soil and freshwater habitats in Austria, People’s Republic of China, Nepal, Uganda, Tanzania, Kenya, New Zealand and Hawaii, and conducted phylogenetic analysis using the small subunit ribosomal RNA (SSU rRNA) gene sequences. We were specifically interested in assessing the diversity of this taxonomically vague group. We hypothesized that (i) the diversity of the ‘Spumella-like’ flagellates is dramatically underestimated by means of conventional light microscopical investigations; (ii) the ‘Spumella-like’ flagellates are not a monophyletic group; and (iii) the ‘Spumella-like’ flagellates do not represent a consistent ecophysiological group. Results Isolation of strains and morphology ‘Spumella-like’ flagellates were present in all of the samples. Successful isolation was, however, hampered in some cases by fast growing bodonids, which in some samples overgrew the colourless chrysophytes. As the ‘Spumella-like’ flagellates could hardly be differentiated by morphological features during the isolation, only one or two, i.e. a small and a large forms, were isolated per sample to avoid multiple isolation of the same clone. All strains possess a spherical to ovate cell body. The strains tended to either attach to the substratum when well fed or to actively swim when starved. Swimming cells tended to spin in small circles even though swimming behaviour differed between the strains. Attached cells did not detach even during cell division, and flagellates were therefore often found in small colonies. All isolated strains were between 3.2 and 8.3 mm long, corresponding to a cell volume of 13–292 mm3 (Table 1). Two unequal flagella inserted close together at the anterior end of the cell, the long flagellum being about 2–4 times longer than the cell body, the short flagellum around 1/2 to 3/4 of the cell diameter and usually of <4 mm length. Only in the strain JBAF35 we found just one flagellum that was visible in the light microscope. The isolates originating from soil (i.e. JBM/S11, JBM/S12, JBC/S23 and JBC/S24) and the freshwater isolate JBC07 had the ability to build cysts, but this ability became weaker in culture and the strains JBC07 and JBC/S23 seemed to have lost the ability to build cysts altogether. Changes in temperature and food conditions did not induce cyst formation in these strains. All strains were bacterivorous and no autofluorescence could be detected. Isolation efficiency For determining the efficiency of the isolation method, i.e. the fraction of flagellate cells which could be successfully isolated in percent of the field abundance of flagellates, filtrated lake water containing the original background bacterial community at in situ temperatures was used. The test on isolation efficiency showed that only 1–2% of the nanoflagellates could be successfully isolated by way of direct dilution. In contrast, after acclimatization of the filtrates, isolation efficiency increased to >80% and was mostly near 100%. It is not clear as to which extent individual cells adapted to the laboratory conditions during this treatment and to which extent more resistant forms replaced the original community. The short acclimatization period of 16–24 h would allow for not more than three to © 2005 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 7, 685–697 Diversity of ‘Spumella-like’ flagellates 687 four subsequent cell divisions only. This implies that a significant fraction of at least 10–20% of the original flagellates were able to individually adapt to the laboratory conditions. Successfully isolated strains could be transferred to a permanent culture with two exceptions: Two isolates could be subcultured at least four times but did finally die back. One of these isolates proved during the isolation process to be a very fast growing flagellate with a generation time of less than 4 h (J. Boenigk, unpublished data). Even different treatments during cultivation such as varying culture media, amounts of wheat grains and bacterial food sources did not result in any permanent culture (J. Boenigk, unpublished data). SSU rRNA gene sequence analysis and phylogenetic affiliation of strains The SSU rRNA gene sequence was determined for 28 ‘Spumella-like’ isolates originating from freshwater and soil habitats in Austria, People’s Republic of China, New Zealand, Nepal, Uganda, Tanzania and Kenya (Table 1). The results of the phylogenetic analysis of the SSU rRNA gene sequences showed that the isolates represent a wide diversity within the chrysophyceae, even though morphological distinction by way of light microscopy was in most cases quite difficult or impossible. Sequence differences among the SSU rRNA gene sequences from our ‘Spumella-like’ flagellates ranged from 0% to 10% (excluding the strains JBAS37 and JBNZ43, which had large A/T-rich insertions). Sequence similarity to the closest-related cultured chrysophyceae was between 92.0% and 99.6% (Table 1). The highest sequence divergence to a database rRNA gene sequence was observed for the strains JBC27, JBM18 and JBM43 (92.0%, 93.9% and 94.3%). In contrast to that, SSU rRNA gene sequences of 50% of the isolates were closely related (i.e. >97% sequence identity) to the known chrysophyceae cultures (Table 1). No correlation could be found between the different regions from which the flagellates were isolated and sequence similarity. Soil as well as aquatic organisms with the same or very similar 18S rRNA gene sequence were isolated from widely different geographic regions. For instance, the soil isolates JBM/S11, JBC/S24 and Spumella elongata showed a sequence similarity of 99.6%, but were isolated from Austria, the People’s Republic of China and the UK (Tables 1 and 2). Similarly, the strains JBM10, JBC07, JBNZ41, JBC30, JBC31 and JBAF32 (sequence identity 100%) originate from locations in Austria, People’s Republic of China, New Zealand and Uganda. The neighbour-joining phylogenetic analysis positioned the isolates in different clusters (Fig. 1); however, bootstrap support (bootstrap value, BV >60%) was observed only for clades A, B1, B2 and E. In general, the clades introduced by Andersen and colleagues (1999) were confirmed by our analysis. The parsimony analysis produced a tree, which recovered all clades, except clade D (Fig. 2). However, the placement of the clades was different and bootstrap support was lower for all of the clades. The majority, i.e. 19 of our isolates affiliated in both analyses with clade C, which however, had no bootstrap support. None of our isolates affiliated with the clade A, which contains the Synurophycean taxa and with clade B1, containing taxa belonging to Hibberdia and related genera (Figs 1 and 2). Sixteen of our clade C isolates grouped within three subclusters of this clade, i.e. the ‘Spumella-like’ soil cluster C1, and the two ‘Spumella-like’ aquatic clusters C2 and C3. Subcluster C2 had different bootstrap values in the distance and the parsimony tree, whereas subcluster C1 showed bootstrap support only in the distance tree. The ‘Spumella-like’ clusters contain exclusively non-green ‘Spumella-like’ isolates, and no green chrysophyceaen strains were affiliated with any of these clusters. All published sequences from isolates originating from soils (Table 2), i.e. the strains Spumella elongata (Belcher and Swale, 1976) and ‘Spumella danica’ (I. Bruchmüller, A. Mylnikov, K. Juergens and T. Weisse, unpublished), were affiliated with cluster C1. Three aquatic isolates (JBAS36, JBM19 and JBC13) were also affiliated with this cluster C1. The aquatic cluster C2 contained both strains from larger lakes, for instance, from Lake Constance (S. obliqua) and Lake Plußsee in Germany (Spumella spp. SpiG, 15G and 37G) (Table 2), and strains from small puddles in Mondsee (JBM09) and Lunz (JBL14) in Austria. All of the new sequences affiliated with the ‘Spumella-like’ cluster C3 had identical 18S rRNA gene sequences and seem to be a sister group to Poterioochromonas spp. A lorica as described for Poterioochromonas spp. has, however, never been observed for new isolates of the cluster C3. In contrast to the distance tree, parsimony analysis indicated that Ochromonas danica and Ochromonas sphaerocystis represent a sister group to C3. Five sequences were related to members of the genus Paraphysomonas. Electron microscopical investigations of these isolates provided evidence that, in contrast to isolates affiliated with the C cluster, all these isolates possessed scales, i.e. morphologically they belong to the genus Paraphysomonas (G. Novarino, pers. comm.). In our analysis bootstrap support did not provide significant measures of confidence that this group is monophyletic. Both analyses resulted in three well-supported lineages within the genus Paraphysomonas, with P. butcheri diverging earlier to the other Paraphysomonas species. The two strains affiliated to the lineage including P. vestita PV10 (Caron et al., 1999) and SOTON1 (Rice et al., 1997), and P. foraminifera HT3, were similar to the two P. vestita © 2005 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 7, 685–697 688 J. Boenigk, K. Pfandl, P. Stadler and A. Chatzinotas Table 1. Origin and characteristics of isolates. Next known sequence Sequence similarity Origin 99.3% Austria, Lake Mondsee JBC07 P. foraminifera TPC2 P. malhamensis 94.9% JBM08 O. tuberculata 95.4% Peoples Republic of China, Lake Tai Hu Austria, Lake Mondsee JBM09 Spumella sp. 15G 99.3% JBM10 P. malhamensis 94.9% JBM/S11 S. elongata 99.6% JBM/S12 S. elongata 98.8% JBC13 ‘S. danica’ 98.9% JBL14 Spumella sp. 15G 99.1% JBM18 O. tuberculata 93.9% JBM19 S. elongata 99.1% JBC22 O. sphaerocystis 97.8% JBC/S23 ‘S. danica’ 98.4% JBC/S24 S. elongata 99.6% JBC27 C. annularis 92.0% JBM28 C. dendrolepidota 95.1% JBC29 95.2% JBC30 P. formamifera SOTON A P. malhamensis JBC31 P. malhamensis 94.9% JBAF32 P. malhamensis 94.9% JBAF33 P. malhamensis 94.9% JBAF35 Oikomonas 98.9% JBAS36 ‘S. danica’ 98.7% JBAS37 P. vestita PV10 99.7% JBNZ39 S. obliqua 97.9% JBNZ40 P. formamifera SOTON A P. malhamensis 95.2% P. vestita SOTON 1 94.3% Isolate JBM06 JBNZ41 JBNZ43 94.9% 94.9% Austria, Puddle in Mondsee Austria, Small artificial pond in Mondsee, Karlsgarten Austria, Soil, Mondsee near ‘Rauchhaus’ Austria, Soil, Mondsee near ‘Rauchhaus’ Peoples Republic of China, Pond in Beijing, Prince Gong’s Mansion Austria, Puddle in Lunz Austria, Lake Krottensee Austria, Lake Hallstatt People’s Republic of China, Pond 1 in Sushou, The Humble Administrator’s Garden People’s Republic of China, Soil near Badaling People’s Republic of China, Soil from Shanghai Peoples Republic of China, Small pond in Huqiu Austria, Lake Schwarzensee People’s Republic of China, Lake Tai Hu People’s Republic of China, Lake Tai Hu Peoples Republic of China, Pond 2 in Sushou, The Humble Administrator’s Garden Uganda, Lake Nkuruba Tanzania, Msimbazi River Kenya, River Sagana Nepal, Nag Pokhari, Kathmandu Nepal, Ranipokhari, Kathmandu New Zealand, Shallow tarn near Karangarua New Zealand, Small lake near Mandeville New Zealand, Lake Aviemore New Zealand, Small stream near Ashburton Latitude longitude Date of isolation Cell size (mm3) 500 17/09/2002 195 ± 63 3 17/11/2002 101 ± 27 500 14/10/2002 55 ± 13 No 500 14/10/2002 85 ± 37 No 500 14/10/2002 34 ± 8 No 47∞52¢0N 13∞20¢60E 47∞52¢0N 13∞20¢60E 39∞53¢60N 116∞24¢46E 500 14/10/2002 36 ± 30 x No 500 14/10/2002 41 ± 18 x No 56 14/11/2002 36 ± 11 No 47∞51¢0N 15∞03¢0E 47∞47¢0 N 13∞23¢20E 47∞32¢60N 13∞39¢0E 31∞18¢28N 120∞37¢10E 884 24/10/2002 135 ± 21 No 580 13/11/2002 35 ± 13 No 556 13/11/2002 16 ± 4 No 3 19/11/2002 61 ± 21 No 40∞20¢15N 115∞58¢10E 795 14/11/2002 13 ± 3 x No 31∞06¢21N 121∞22¢31E 5 21/11/2002 15 ± 6 x No 31∞20¢05N 120∞34¢27E 4 20/11/2002 205 ± 22 No 47∞45¢0N 13∞29¢50E 31∞30¢0N 120∞20¢0E 31∞30¢0N 120∞20¢0E 31∞18¢28N 120∞37¢10E 716 13/11/2002 31 ± 12 No 3 17/11/2002 58 ± 18 No 3 19/11/2002 28 ± 10 No 3 19/11/2002 27 ± 9 No 0∞37¢0N 30∞16¢0E 5∞15¢0S 38∞49¢60E 0∞40¢0S 37∞12¢0E 27∞43¢0N 85∞19¢0E 27∞43¢0N 85∞19¢0E 43∞37¢0S 169∞46¢0E 1400 22/03/2003 66 ± 23 No 151 20/03/2003 68 ± 20 No 1207 28/03/2003 58 ± 13 No 1298 27/03/2003 35 ± 17 No 1298 30/03/2003 202 ± 52 No 1118 01/02/2003 25 ± 8 No 46∞0¢0S 168∞49¢0E 44∞40¢60S 170∞22¢0E 43∞58¢0S 171∞46¢0E 104 02/02/2003 119 ± 26 No 212 03/02/2003 62 ± 11 No 60 05/02/2003 292 ± 129 No 47∞52¢0N 13∞20¢60E 31∞30¢0N 120∞20¢0E 47∞52¢0N 13∞20¢60E 47∞52¢0N 13∞20¢60E 47∞52¢0N 13∞20¢60E Altitude (m) Cysts Autofluorescence No x No © 2005 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 7, 685–697 Diversity of ‘Spumella-like’ flagellates 689 Table 2. Known sequences of clade C of the Chrysophyceae (18S rRNA gene) following Andersen and colleagues (1999). The origin of the strains and the GenBank entry number are shown. Strain name Origin of strain and identification number GenBank entry Reference Spumella danica nov. sp. Jutland, Denmark: soil AJ236861 Spumella elongata Type strain Girton, Cambridgeshire, UK: soil. CCAP strain no. 955/1 Baden-Württemberg, Germany: Lake Constance – freshwater Schleswig-Holstein, Germany: Lake Behler See – freshwater Schleswig-Holstein, Germany: Pond near Plön – freshwater Schleswig-Holstein, Germany: Lake Plußsee – freshwater AJ236859 I. Bruchmüller, A. Mylnikov, K. Juergens and T. Weisse, unpublished Bruchmüller (1998) Freshwater MCC-NIES strain MBI HT2 (no longer available) Michigan, USA: roadside ditch – freshwater CCMP strain 1862 Port Phillip Bay, Melbourne, Australia: marine CCMP strain 1278 Sargasso Sea: marine CCMP strain 584 Type strain Everdrup, Denmark: Bog-pool – freshwater. UTEX strain 1298 Arkansas, USA: small stream – freshwater CCMP strain 586 Illinois, USA: Volo Bog – freshwater CCMP strain 1861 Alberta, Canada: Glenmore reservoir – freshwater CCMP strain 1863 Washington, USA: North Atlantic – marine CCMP strain 591 Maine, USA: west Boothbay Harbour – marine CCMP strain 1860 Alberta, Canada: Glenmore reservoir – freshwater CCMP strain 1859 Tasmania, Australia: Golden Cloud Swamp – freshwater AB02307 Bruchmüller (1998) Bruchmüller (1998) Bruchmüller (1998) Synonym to Spumella sp. SpG Bruchmüller (1998) Andersen et al. (1999) AF123295 Andersen et al. (1999) U42382 Andersen et al. (1999) U42381 Andersen et al. (1999) M32704 Gunderson et al. (1987) AF123294 Andersen et al. (1999) AF123293 Andersen et al. (1999) AF123290 Andersen et al. (1999) AF123302 Andersen et al. (1999) AF123291 Andersen et al. (1999) AF123302 Andersen et al. (1999) U71196 Saunders et al. (1997) AF123297 Andersen et al. (1999) AF123298 Andersen et al. (1999) AF123301 Andersen et al. (1999) Spumella Spumella Spumella Spumella obliqua sp. 15G sp. 37G sp. SpiG Poterioochromonas malhamensis Poterioochromonas stipitata Ochromonas CCMP1278 Ochromonas CCMP 584 Ochromonas danica Ochromonas sphaerocystis Ochromonas tuberculata Uroglena americana Chrysoxis sp. Dinobryon sociale var. americana Dinobryon sertularia Chrysonephele palustris Chrysolepidomonas dendrolepidota Epipyxis aurea Epipyxis pulchra Michigan, USA: Lake Medora – freshwater CCMP strain 293 Minnesota, USA: Darling Pond – freshwater CCMP strain 385 Minnesota, USA: Darling Pond – freshwater CCMP strain 382 AJ236860 AJ236857 AJ236858 AJ236862 CCAP: Culture Collection of Algae and Protozoa UK; CCMP: Provasoli – Guillard National Centre for Culture of Marine Phytoplankton; MCCNIES: Microbial Culture Collection at the national institute for environmental studies; UTEX: University of Texas, Culture Collection of Algae. sequences characterized by the A/T-rich insertion sequences. The largest A/T-rich insertions as found in P. vestita PV10 were identically present in JBAS37. Discussion Older systems of classification have been based on vegetative morphological features and characteristics of the motile cell, especially the flagellar number and position (Preisig, 1995). Even though the flagellar number has been disregarded as a major taxonomic criterion (Kristiansen, 1986, 1990) it is still used for separating genera. Members of the chrysophycean order Chromulinales (following Preisig, 1995) possess two flagella where the second flagellum is short in Chromulina spp. and Oikomonas spp. and long in Ochromonas spp. and Spumella spp. Except for this difference Ochromonas and Chromulina on the one hand, and Spumella and Oikomonas on the other are similar (Preisig, 1995). All of our isolates possessed a spherical to ellipsoidal cell body and attached to the substrate when satiated. Most strains possessed two flagella, a long and a short one, emerging from the anterior end. The short flagellum was several micrometres in length and following the light microscopical and electron microscopical investigations the isolates belong to the Spumella/Monas group. The strains specifically affiliated with cluster F possess scales and thus belong to the genus Paraphysomonas (G. Novarino, pers. comm.). Only in the strain JBAF35 was the short flagellum not visible in the light microscope, or extremely short, and the strains may therefore be affiliated with Oikomonas. This © 2005 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 7, 685–697 690 J. Boenigk, K. Pfandl, P. Stadler and A. Chatzinotas Paraphysomonas butcheri Paraphysomonas bandaiensis Paraphysomonas foraminifera SOTON A Paraphysomonas foraminifera TPC2 Paraphysomonas imperforata VS1 96 98 61 81 JBM06 Paraphysomonas sp. HD 100 Paraphysomonas vestita SOTON 1 Paraphysomonas foraminifera HT3 61 100 65 F JBNZ43 100 Paraphysomonas vestita PV10 JBAS37 100 JBNZ40 JBC29 100 66 66 97 66 Ochromonas tuberculata Chromulina chionophila Chrysosaccus sp. CCMP295-II Chrysosaccus sp. CCMP295-I Chrysosphaera parvula Lagynion scherffelii 100 JBM08 JBM18 JBM28 95 97 E Ochromonas danica Ochromonas sphaerocystis JBC22 JBAF33 JBAF32 100 JBM10 JBC07 JBNZ41 JBC30 JBC31 81 C3 Poterioochromonas malhamensis Poterioochromonas stipitata Chrysonephele palustris Epipyxis aurea Epipyxis pulchra 100 Dinobryon sociale Dinobryon sertularia Ochromonas sp. CCMP1278 Chrysoxys sp. CCMP591 100 96 Fig. 1. Neighbour-joining tree showing the affiliation of 18S rRNA gene sequences from ‘Spumella-like’ isolates to the Chrysophyceae sensu stricto. The numbers at the nodes of the tree indicate percentage of bootstrap values for each node out of 100 bootstrap resamplings (values above 60 are shown). The scale bar indicates 2% estimated sequence divergence. C JBC/S23 Spumella danica 99 JBAS36 JBC13 96 Spumella elongata 70 JBC/S24 C1 JBM/S11 JBM/S12 JBM19 Uroglena americana Ochromonas sp. CCMP584 Chrysolepidomonas dendrolepidota 100 Spumella obliqua JBNZ39 66 85 84 70 92 Spumella sp. SpiG Spumella sp. 15G Spumella sp. 37G JBL14 JBM09 C2 Phaeoplaca thallosa Cyclonexis annularis JBC27 100 87 84 Tessellaria volvocina Synura sphagnicola 100 Synura petersenii Synura glabra Synura uvella Synura spinosa Synura mammillosa 100 Mallomonas rasilis Mallomonas papillosa Mallomonas akrokomos Mallomonas annulata 100 Mallomonas caudata Mallomonas matvienkoae 92 Mallomonas striata Mallomonas striata MUC295 92 Mallomonas splendens Mallomonas adamas Chromulina nebulosa 100 Chrysamoeba mikrokonta Chrysamoeba pyrenoidifera 100 Oikomonas mutabilis D A B2 JBAF35 Chrysocapsa vernalis Hibberdia magna Chromophyton rosanoffii Chrysochaete britannica Nannochloropsis granulata 97 B1 2% © 2005 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 7, 685–697 Diversity of ‘Spumella-like’ flagellates 691 Tessellaria volvocina Synura glabra Synura petersenii Mallomonas rasilis 99 Mallomonas papillosa Mallomonas akrokomos Mallomonas annulata 99 Mallomonas matvienkoae Mallomonas caudata 66 Mallomonas striata MUC295 Mallomonas striata 95 Mallomonas splendens 67 Mallomonas adamas Synura uvella Synura mammillosa Synura spinosa Synura sphagnicola Paraphysomonas butcheri Paraphysomonas bandaiensis 74 Paraphysomonas foraminifera SOTON A 94 Paraphysomonas foraminifera TPC2 Paraphysomonas imperforata VS1 60 JBM06 Paraphysomonas sp. HD 99 JBC29 JBNZ40 JBNZ43 99 Paraphysomonas foraminifera HT3 99 Paraphysomonas vestita SOTON 1 99 Paraphysomonas vestita PV10 JBAS37 Phaeoplaca thallosa Ochromonas tuberculata 64 Chromulina chionophila 91 99 Chrysosaccus sp. CCMP295-II 98 Chrysosaccus sp. CCMP295-I 69 Chrysophaera parvula Lagynion scherffelii JBM08 JBM18 JBAF33 Ochromonas danica 98 99 Ochromonas sphaerocystis JBC22 JBC31 67 JBC30 82 64 JBC07 99 JBM10 JBAF32 JBNZ41 99 Poterioochromonas malhamensis Poterioochromonas stipitata 99 Dinobryon sociale Dinobryon sertularia 92 Ochromonas sp. CCMP1278 Chrysoxys sp. CCMP591 JBC/S23 Spumella danica JBAS36 JBC13 Spumella elongata 89 JBC/S24 93 JBM/S11 JBM/S12 JBM19 Uroglena americana Ochromonas sp. CCMP 584 99 Chrysonephele palustris Epipyxis aurea Epipyxis pulchra Chrysolepidomonas dendrolepidota JBM28 Spumella obliqua 99 JBNZ39 83 Spumellla sp. SpiG Spumella sp. 37G Spumella sp. 15G 63 JBL14 JBM09 Chromulina nebulosa 75 99 Chrysamoeba mikrokonta Chrysamoeba pyrenoidifera 99 Oikomonas mutabilis JBAF35 82 Cyclonexis annularis JBC27 Chrysocapsa vernalis 94 Hibberdia magna 70 Chromophyton rosanoffii 74 Chrysochaete britannica Nannochloropsis granulata 99 A Fig. 2. Parsimony tree showing the affiliation of 18S rRNA gene sequences from ‘Spumella-like’ isolates to the Chrysophyceae sensu stricto. The numbers at the nodes of the tree indicate percentage of bootstrap values for each node out of 100 bootstrap resamplings (values above 60 are shown). F E C3 C C1 C2 conclusion seems to be supported by the high 18S rRNA gene sequence similarity of JBAF35 and Oikomonas mutabilis. We observed, however, no consistent trend in length and visibility of the short flagellum, and a distinction between Spumella and Oikomonas based on only this character may be problematic. In general, the lack of distinctive characters in the ‘Spumella-like’ flagellates, as discussed above, may not allow for the separation of taxa based solely on morphological characters. B2 B1 Some prominent genera within the chrysophytes seem to be polyphyletic In agreement with the study of Andersen and colleagues (1999) we found indications that Ochromonas is polyphyletic and occupied different branches within clade C and clade E. Polyphylie can also be expected for the colourless analogue of Ochromonas, i.e. Spumella. In fact, we found a high 18S rRNA gene diversity of the © 2005 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 7, 685–697 692 J. Boenigk, K. Pfandl, P. Stadler and A. Chatzinotas ‘Spumella-like’ flagellates, which grouped within different clades in the class Chrysophyceae sensu stricto (Andersen et al., 1999). Isolates affiliated with the cluster F belong to the genus Paraphysomonas as they possess scales (G. Novarino pers. comm.). The genus Spumella, however, is polyphyletic and distributed throughout at least three subclusters which are characterized by isolates of different habitat types. All isolates from soil were affiliated with cluster C1. There are also three aquatic isolates grouping within this cluster, but we cannot exclude the possibility that these strains were introduced into the aquatic environment from the surrounding soils. Further studies are required to prove that SSU rRNA gene sequences are a useful marker to distinguish ‘Spumella-like’ soil flagellates from aquatic strains. Similarly, it may be possible to attribute some rough ecological characteristics to the subcluster C3 strains. Most strains of the C3 cluster originated from shallow, often eutrophic habitats and our attempts to obtain axenic flagellate cultures succeeded only with isolates from this cluster (J. Boenigk, unpublished data). This suggests that some essential factors can be synthesized by these isolates in laboratory cultures. Specific nutritive requirements of chrysophytes are already indicated for the mixotrophic genera Ochromonas and Poterioochromonas (cf. Holen and Boraas, 1995). The ‘Spumella-like’ flagellates are a diverse group both in terms of sequence dissimilarity between isolates and in terms of the number of genotypes The phylogenetic distance to known sequences was high for many of our isolates and thus the ‘Spumella-like’ flagellates seem to be a very diverse group. Isolation attempts from samples obtained from 24 ecologically contrasting freshwater and soil habitats located in six climatic zones resulted in 28 isolates distributed over the entire SSU rRNA tree of the Chrysophyceae sensu stricto. Some of these isolates, e.g. JBM18 probably represent even novel lineages within this group, indicating that analysis of further isolates is necessary to support these lineages. Furthermore, we probably even underestimated the diversity of these flagellates, as the isolation approach we used in our study must be assumed to be selective: isolation efficiency using direct dilution of environmental samples was as low as 1–2% only, but could be increased to >10–20% by way of short-term acclimatization. The isolation procedure we used selected for small bacterivorous flagellates with medium to high growth rates, probably resulting in the isolation of only the fast-growing fraction of the ‘Spumella-like’ organisms. Long-term controls confirmed that the positive growth of these flagellates could be definitely detected within the subculturing periods of 5–7 days and it can be assumed that at least these acclimatized flagellates in particular were the only fast-growing strains. Except the bias resulting from the initial treatment, i.e. cell losses because of sampling and shifts during the initial acclimatization period, a further bias resulting from the isolation protocol is therefore assumed to be low. The literature data on isolation efficiency are rare, but low isolation efficiency has also been observed by other researchers (G. Novarino, pers. comm.). Low efficiency of isolation is also supported by the contrasting high diversity of free-living protists and the relatively small group of protists successfully cultured in the laboratory (cf. Cowling, 1991). Recently, culture-independent molecular surveys on eukaryotic diversity in marine systems suggest that, there still is undescribed protistan diversity, particularly among small eukaryotic organisms (Moreira and Lopez-Garcia, 2002 and references therein). As isolation procedures often include an acclimatization period (Caron, 1993; Atkins et al., 2000; Lim et al., 2001), the dominance of the so-called ‘laboratory weeds’ do not, however, necessarily reflect a differential ability to grow in culture, but may indicate that these usually fast-growing organisms overgrow other protists during this acclimatization and consequently are isolated more often. The diversity of the ‘Spumella-like’ flagellates must therefore be assumed to be even higher than is reported in this study. Given the fact that such a diverse group as the ‘Spumella-like’ flagellates, which based on our sequence data probably comprises different independent lineages, is usually pooled in field investigations it is not surprising that correlations with habitat characteristics and environmental factors are often weak. Organisms affiliated with the ‘Spumella-like’ clusters C1 through C3 seem to be distributed worldwide but ecophysiological characteristics seem to differ Diversity of protists and protist species richness are currently controversially discussed, mainly based on organisms possessing suitable morphological features, such as ciliates and the colourless chrysophyte genus Paraphysomonas (Finlay and Clarke, 1999a,b; Finlay and Fenchel, 1999; Foissner, 1999; Finlay, 2002). For instance, specific protistan morphotypes and even the same genotypes have been reported from different locations all over the world (Finlay, 2002). For the ‘Spumellalike’ flagellates, because of the scarcity of both morphological features and molecular data, estimates of the diversity and their biogeographic distribution are even more complicated and it has even been proposed that most described species may belong to other taxa (Preisig et al., 1991). Consequently, the ‘Spumella-like’ flagellates are mostly pooled in ecological studies (‘Spumella-like’ flagellates or Spumella spp.) and this © 2005 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 7, 685–697 Diversity of ‘Spumella-like’ flagellates 693 group often comprises some Paraphysomonas spp. as well, because these two groups cannot sufficiently be separated by light microscopy (e.g. Weitere and Arndt, 2003). These ‘Spumella-like’ flagellates are present in most ecosystems and are of general importance as one of the main eukaryotic heterotrophs in a variety of ecosystems often accounting for 20–50% of pelagic heterotrophic nanoflagellates (Carrick and Fahnenstiel, 1989; Sanders et al., 1989; Bennet et al., 1990; Carrias et al., 1998; Arndt et al., 2000; Weitere and Arndt, 2003). The ‘Spumella-like’ flagellates therefore are regarded as a ubiquitous, worldwide-distributed group commonly found in freshwaters, soils and marine sites (Preisig et al., 1991). Accordingly, we detected ‘Spumella-like’ organisms in all of the samples. In the 5 mm fraction these organisms were among the dominant organisms in most of the processed samples. Despite the lack of morphological distinctive characteristics we found evidence for a global distribution of closely related strains: For instance, the same genotype in terms of the 18S rRNA gene sequence similarity was successfully isolated from freshwater habitats located in Austria, China, New Zealand and East Africa, and very similar genotypes (>99.6% 18S rRNA gene sequence similarity) were obtained from soil habitats located in Austria, the UK and China. On the basis of the 18S rRNA gene sequence similarity, our findings may support the hypothesis of a worldwide distribution of microbial species (Finlay, 2002). Identical and very similar 18S rRNA sequences for eukaryotic microorganisms originating from geographically distant sites have been reported also for marine flagellates and marine clone sequences (Atkins et al., 2000; Massana et al., 2004). Although multiple rRNA gene copies with sequence variations seem to be present among different taxa of eukaryotic microorganisms (Ward et al., 1997; Rocio et al., 1998), we could not find any. However, eukaryotic microorganisms with very similar or identical rRNA gene sequence may show more differences in the more variable internal transcribed spacer (ITS) region (Tsuchiya et al., 2003). It remains to be evaluated if ITS-sequences may also aid in revealing cryptic speciation in heterotrophic nanoflagellates. However, despite the high 18S rRNA gene sequence similarities of strains from geographically remote sampling sites, there are indications for ecological differences between these isolates. For instance, the isolates JBC07, JBM10 and JBNZ41 (18S rRNA gene sequence similarity 100%), respond differentially to ecological factors such as food concentration, food quality and suspended sediment concentrations (Boenigk et al., 2004; K. Pfandl, J. Boenigk and A. Wiedlroither, in prep.). These findings corroborate the idea that molecular data on the basis of the 18S rRNA gene may not suffice in separating ecologically different taxa and ecotypes. The above rough trends concerning ecological key parameters should therefore be interpreted carefully. Conclusions On the basis of these 18S rRNA gene sequence data, global distribution of the ‘Spumella-like’ flagellates must be assumed, but there are indications that even closely related isolates show different ecophysiological abilities, which may restrict these organisms to a certain habitat or geographical region. Following our phylogenetic analysis we conclude that: (i) the ‘Spumella-like’ flagellates are a very diverse group, and deduced from the relatively high phylogenetic distance of many isolates to the next known organisms, diversity seems to still be strongly underestimated; (ii) several chrysophycean genera including Ochromonas and Spumella are polyphyletic; and (iii) based on the SSU rRNA gene sequence there are thus far no indications for a geographic restriction, but there may be different ecophysiological abilities characterizing certain branches of ‘Spumella-like’ flagellates. Experimental procedures Sampling sites Samples from 24 ecologically contrasting freshwater and soil habitats located in six climatic zones were processed in order to isolate representative ‘Spumella-like’ flagellates (Table 1). Samples taken from Lake Mondsee and sampling sites in Mondsee were immediately processed after sampling. Samples from the other lakes in the Salzkammergut area were processed within 6 h after sampling. All other samples were transported to the laboratory in sealed tubes and processed within a few days after sampling. All treatments after sampling were carried out under aseptic conditions. Media for isolation and the maintenance of strains Standard medium for the maintenance of strains and for the isolation process, except for the first step, was an artificial inorganic basal medium (NSY medium: Hahn et al., 2003). Sterile-filtrated lake water for the first isolation step was prepared using water sampled from a depth of 1 m in Lake Mondsee. Water was filtrated through 0.1 mm gauze and subsequently repeatedly heated in the microwave for 7.5 min at 700 W. This procedure killed all of the bacteria that might have been present in the filtrate. To obtain lake water containing a natural bacterial assemblage as a food source, the lake water was filtered through 1.2 mm filters. This filtrate contained 2–3 ¥ 106 bacteria per millilitre. Different bacterial strains, representatives of typical freeliving aquatic bacteria and a natural bacterial community were used as food for isolating flagellates: The ultramicrobacterial actinobacterium strain (<0.1 mm3) MWH-Mo1 [closest known relative Clavibacter michiganensis (Microbacteriaceae); Hahn et al., 2003], the bacterial strain Listonella pelagia CB5 (Hahn, 1997), and a natural bacterial © 2005 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 7, 685–697 694 J. Boenigk, K. Pfandl, P. Stadler and A. Chatzinotas community from Lake Mondsee sampled from a depth of 1 m (for isolation of strains from Lake Mondsee only). The filtrate was subsequently checked for flagellate contamination. After successful isolation of the flagellate strains, they were transferred to permanent culture using NSY medium supplemented with wheat grain. Isolation protocol A modified filtration–acclimatization method (Hahn et al., 2004) adjusted for the isolation of flagellates was used. Briefly, 5–20 ml of the sample was filtered through 5 mmpore-size filters (Minisart syringe filters; Sartorius, Göttingen, Germany) and collected in sterile Erlenmeyer flasks. After 16–24 h of acclimatization at 15–25∞C depending on the origin of the samples, subsamples of 5 ml were stepwise diluted (three subsequent 1:1 dilutions) with sterile-filtrated lake water. After 2–4 h the flagellates were counted using a Sedgewick-Rafter chamber, and a subsample was diluted into a final flagellate abundance of 0.5–1 flagellates per millilitre and subsequently transferred to two to four 24-well cell culture plates. Wells were supplemented with food bacteria at a concentration of 3–5 ¥ 106 bacteria per millilitre, i.e. either a 1.2 mm filtrate of freshly sampled lake water (only for samples from Lake Mondsee), or cultures of the bacterial strains MWH-Mo1 or L. pelagia CB5. Wells were checked every second day for a period of at least 2 weeks for positive growth under the microscope using a total magnification of 200¥. When flagellate growth was detected, the medium was transferred to a 50 ml Erlenmeyer flask containing inorganic NSY medium and fresh food bacteria. After 2–6 days the subsamples were further diluted to a final concentration of 0.05, 0.1, 0.2 and 0.4 flagellates per millilitre and supplemented with the bacterial strains MWHMo1 or CB5 at a concentration of 15–25 ¥ 106 bacteria per millilitre. Each of these dilutions were transferred to wells of sterile 24-well cell culture plates (1 ml per well) and incubated at 15–22∞C depending on the origin of the isolates. Screening of the wells for the growth of flagellates was again performed by direct microscopical investigation every second day. Finally, flagellates were transferred to an Erlenmeyer flask containing fresh medium and bacteria. This procedure was repeated until pure cultures were established, but at least four times. Pure cultures were acclimatized to 15∞C and transferred to permanent culture. Isolation efficiency The cultivable fraction of HNF was estimated by way of the direct dilution of freshwater samples. This test was only performed exemplarily for four samples from Lake Mondsee. Flagellates in the samples were counted using a live counting technique in a Sedgewick-Rafter chamber. A subsample was then diluted to a concentration of 0.5–1 flagellate per millilitre using 1.2 mm filtrated water from the same sample. Flagellate abundance was again checked and 1 ml was transferred to each well of 24-well cell culture plates and incubated at an in situ temperature. Altogether 16 plates were used per sample. The wells were checked microscopically for flagellates every two to three days for a period of at least 30 days. DNA extraction and PCR of 18S rRNA genes DNA was isolated from cell pellets or 0.5 ml cell cultures. Cells were incubated with 1.5 ml extraction buffer (10 mM Tris-HCl, 0.4% SDS, 10 mM EDTA) at 65∞C, followed by incubation with proteinase K at 60∞C (0.2 mg ml-1). DNA was pelleted with ethanol after phenol-chloroform extraction. Small subunit ribosomal RNA genes were amplified using one of the two forward primers CTGGTTGATCCTGCCAG (Paraphysomonas foraminifera AB022864 position 30) and GAAACTGCGAATGGCTC (P. foraminifera position 109), and one of the two reverse primers GTAGGTGAACCTGCAG (P. foraminifera position 1824) and GTGAACCTGCAGAAG GATCA (P. foraminifera position 1828). The polymerase chain reaction (PCR) mixture contained 0.2 mM of each primer, 200 mM of each deoxynucleoside triphosphate, 2 mM MgCl2, 1.25 U of Taq DNA polymerase (Qiagen), and 1¥ PCR buffer. Reactions were carried out in a PTC 200 thermocycler (MJ Research) starting with a denaturation at 94∞C for 3 min, followed by 30 cycles of denaturation at 94∞C for 45 s, annealing at 58∞C for 1 min, elongation at 72∞C for 2 min, and a final extension step of 10 min at 72∞C. PCR products were checked on an agarose gel. Sequencing of 18S rRNA genes and phylogenetic analysis Polymerase chain reaction products were either directly sequenced or used for subsequent cloning into the vector pGEM-T Easy (Promega) by following the manufacturer’s recommendations. Putative positive colonies were picked and directly amplified, using the vector primers T7 and SP6. The products were checked on an agarose gel. Sequencing reactions were performed with an ABI Prism® Big Dye™ Terminator v 3.0 Ready Reaction Cycle Sequencing Kit (Applied Biosciences) and an ABI PRISM model 3100 automated sequencer. Sequences were submitted to the BLAST search program of the National Center for Biotechnology Information (NCBI) to find closely related sequences. Sequences were aligned using the ‘CLUSTAL W’ option (Thompson et al., 1997) in the BioEdit 5.0.9 sequence analysis software (Hall, 1999). Where necessary alignments were subsequently manually processed and corrected. Positions of unclear homology and AT-rich insertions were excluded from further phylogenetic analysis. The result was a final alignment of 1630 positions (including gaps). Sequence identities between isolates and the next related sequences were based on aligned whole gene sequences of equal length, and were calculated using the ‘sequence identity matrix’ option in BioEdit. The position of the unclear homology and A/T-rich insertions were excluded from further phylogenetic analysis. The TREECON 1.3b software package was used to calculate distance matrices by the Kimura algorithm (Kimura, 1980) and to generate phylogenetic trees by the neighbour-joining method (van de Peer and de Wachter, 1994). Parsimony trees were calculated using the program package PHYLIP (version 3.5; J. Felsenstein, Department of Genetics, University of Washington, Seattle). One hundred bootstrapped replicate resampling data were generated with SEQBOOT (PHYLIP). The full-length 18S rRNA gene sequences determined in this study have a length of 1695–1803 bp and have © 2005 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 7, 685–697 Diversity of ‘Spumella-like’ flagellates 695 been deposited in the NCBI database under accession numbers AY651071–651098. Cell size and autofluorescence Live cells from early stationary growth phase were taken for measuring cell dimensions. Cells were transferred to observation vessels (Boenigk and Arndt, 2000) and recorded on video tape using 500¥ to 1000¥ magnification. The length and width of at least 30 cells was measured directly from the video screen and the cell volume was calculated assuming an ellipsoid or a spherical cell shape. Cells were also checked for the occurrence of chloroplasts. In addition, cells were checked for autofluorescence: Cells were fixed with formaldehyde (final concentration 2%) and stained with DAPI (final concentration 10 mg ml-1) for 30 min. The cells were then filtered onto a black nucleopore 0.2 mm filter backed by a 0.45 mm cellulose nitrate filter and examined under an epifluorescence microscope using UV and blue light excitation for DAPI and for chlorophyll autofluorescence respectively. Acknowledgements The authors would like to thank M. 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